Stabilization of E. coli Ribonuclease HI by the ‘stability profile of mutant protein’ (SPMP)-inspired random and non-random mutagenesis

The change in the structural stability of Escherichia coli ribonuclease HI (RNase HI) due to single amino acid substitutions has been estimated computationally by the stability profile of mutant protein (SPMP) [Ota, M., Kanaya, S. Nishikawa, K., 1995. Desk-top analysis of the structural stability of various point mutations introduced into ribonuclease H. J. Mol. Biol. 248, 733–738]. As well, an effective strategy using random mutagenesis and genetic selection has been developed to obtain E. coli RNase HI mutants with enhanced thermostability [Haruki, M., Noguchi, E., Akasako, A., Oobatake, M., Itaya, M., Kanaya, S., 1994. A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions. J. Biol. Chem. 269, 26904–26911]. In this study, both methods were combined: random mutations were individually introduced to Lys99–Val101 on the N-terminus of the α-helix IV and the preceding β-turn, where substitutions of other amino acid residues were expected to significantly increase the stability from SPMP, and then followed by genetic selection. Val101 to Ala, Gln, and Arg mutations were selected by genetic selection. The Val101→Ala mutation increased the thermal stability of E. coli RNase HI by 2.0 °C in Tm at pH 5.5, whereas the Val101→Gln and Val101→Arg mutations decreased the thermostability. Separately, the Lys99→Pro and Asn100→Gly mutations were also introduced directly. The Lys99→Pro mutation increased the thermostability of E. coli RNase HI by 1.8 °C in Tm at pH 5.5, whereas the Asn100→Gly mutation decreased the thermostability by 17 °C. In addition, the Lys99→Pro mutation altered the dependence of the enzymatic activity on divalent metal ions.